Polymer blends as biodegradable matrices for preparing biocomposites

ABSTRACT

The present invention provides bioerodable constructs for controlled release of bioactive materials. In a preferred mode, the constructs may be utilized adjacent to a biological surface. The constructs are based on a blend of two or more poly(ester-amide) polymers (PEA). Such polymers may be prepared by polymerization of a diol (D), a dicarboxylic acid (C) and an alpha-amino acid (A) through ester and amide links in the form (DACA) n . An example of a (DACA) n  polymer is shown below in formula II. Suitable amino acids include any natural or synthetic alpha-amino acid, preferably neutral amino acids.

This application claims benefit of Provisional Application No.60/175,377 filed on Jan. 11, 2000 and Provisional No. 60/175,415, filedJan. 11, 2000 and Provisional No. 60/175,416 filed Jan. 11, 2000 andProvisional No. 60/205, 240 filed on May 19, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to polymeric matrices designed for controlledrelease of biologically active substances, such as therapeuticbacteriophage which can kill bacteria capable of causing disease.

2. Review of Related Art

Bioactive composites based on biodegradable (or more precisely,bioerodible) polymers as matrices, impregnated by bactericidalsubstances are promising for the treatment of superficial infectedwounds. On the one hand, bactericidal substances clean the wound frombacteria and make favorable conditions for wound healing, and preventbacterial invasion through the holes made in wound coverings for exudatedrainage, on drainage. On the other hand, biodegradable polymer which isable to timely release enough degradation products (polymeric debris)can activate macrophages to produce the required growth factors acrdand, in that way, can accelerate wound healing (Pratt, et al. (1994,“Dimehtyltitanocene-Induced Surface Chemical Degradation of SyntheticBioabsorbable Polyesters”, J. Polym. Sci. Part 0.4: Polym. Chem.,32(5):949; Greisler, (1988), “Small Diameter Vascular Prostheses:Macrophage-Biomaterial Interactions with Bioresorbable VascularProstheses”. Transactions of ASAIO, 34:1051).

Mori, et al., U.S. Pat. No. 3,867, 520, discloses a delivery system fortherapeutic agents using films made of polyamino acid polymers withoil-like or wax-like substances dispersed in the film. Therapeuticagents are dissolved in the carrier, and when the film is applied to aninternal or external surface of the body, the carrier migrates to thesurface of the film where the agent is released. However, these filmsare not biodegraded during use.

Sidman, U.S. Pat. No. 4,351,337, discloses an implantable deliverydevice comprising a matrix formed of a poly-alpha-amino acid componenthaving one or more drugs and/or diagnostic agents physically containedtherein. The drug or diagnostic agent is released through diffusionand/or biodegradation resulting from the action on the polymeric matrixof enzymes present in the host into which the implant is placed.

Taniharak, et al., U.S. Pat. No. 5,770,229, discloses a medical polymergel made up of a cross-linked polysaccharide with a drug attackedattached to the polysaccharide via a linkage that is cleavable by anendogenous enzyme. This system provides for delayed release of theattached drug from the polymer, but the release rate is subject toindividual variation in the amount of the endogenous enzyme, and thepolymer, while biocompatible, is not biodegradable.

Kuroyangi and coworkers (1992, J. Appl. Biomater., 3:153-161) havedeveloped a wound dressing for burn care that is a hydrophobicpoly-L-leucine spongy matrix impregnated with antibacterial silversulfadiazine supported by a fine nylon mesh. This wound dressingsuppresses bacterial growth while controlling fluid loss. However, thedressing is not degraded, but rather sticks to the wound until itseparates spontaneously from the healed skin.

Georgian Patent No. 1090 describes a wound dressing containing 45-50 wt.% biodegradable poly(ester-amide) based on natural alpha-amino acidsimpregnated with 50-55 wt. % dried bacteriophage. The poly(ester-amide)is not characterized in detail, but the dressing also has 0.05-0.15 wt.% surface immobilized alpha-chymotrypsin. The impregnatedpoly(ester-amide) is formed into a film, and the film is used toaccelerate healing of superficial wounds, including burns.

Tsitlanadze, et al., in an abstract from Int. Symp. Biodegrad. Mater,Oct. 7-9, 1996, Hamburg, Germany, describe alpha-chymotrypsin-catalyzedhydrolysis of regular poly (ester-amides) (PEAs) of general formula I:

where

-   -   k=2, 3, 4, or 6    -   m=4 or 8, and    -   R=CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, (CH₂)₃CH₃, CH₂C₆H₅, or        (CH₂)₃SCH₃.

It is reported that alpha-chymotrypsin is spontaneously immobilized onthe surface of the PEAs from aqueous solution, and erodes the polymersurface under physiologic conditions, with increasing lysis for morehydrophobic R groups and more hydrophobic polymer backbone. Abiocomposite material based on a PEA polymer containing bacteriophages,antibiotic or anesthetic was prepared for study as artificial skin forhealing burns and festering wounds.

SUMMARY OF THE INVENTION

The present invention provides bioerodable constructs for controlledrelease of bioactive materials. In a preferred mode, the constructs maybe utilized adjacent to a biological surface. The constructs are basedon a blend of two or more poly(ester-amide) polymers (PEA). Suchpolymers may be prepared by polymerization of diol (D), a dicarboxylicacid (C) and an alpha-amino acid (A) through ester and amide links inthe form (DACA)_(n). An example of a (DACA)_(n) polymer is shown belowin formula II. Suitable amino acids include any natural or syntheticalpha-amino acid, preferably neutral amino acids.

Diols may be any aliphatic diol, including alkylene diols likeHO—(CH₂)_(k)—OH (i.e. non-branched), branched diols (e.g., propyleneglycol), cyclic diols (e.g. dianhydrohexitols and cyclohexanediol), oroligomeric diols based on ethylene glycol (e.g., diethylene glycol,triethylene glycol, tetraethylene glycol, or poly(ethylene glycol)s).Aromatic diols (e.g. bis-phenols) are less useful for these purposessince they are more toxic, and polymers based on them have rigid chainsthat are less likely to biodegrade.

Dicarboxylic acids may be any aliphatic dicarboxylic acid, such asα,ω-dicarboxylic acids (i.e., non-branched), branched dicarboxylicacids, cyclic dicarboxylic acids (e.g. cyclohexanedicarboxylic acid).Aromatic diacids (like phthalic acids, etc.) are less useful for thesepurposes since they are more toxic, and polymers based on them haverigid chain structure, exhibit poorer film-forming properties and havemuch lower tendency to biodegrade.

Preferred PEA polymers have the formula II:

where

-   -   k=2-12, especially 2, 3, 4, or 6,    -   m=2-12, especially 4 or 8, and    -   R=CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, (CH₂)₃CH₃, CH₂C₆H₅, or        (CH₂)₃SCH₃ (CH₂ ) ₂ SCH ₃.

The constructs optionally contain bioactive inclusions, which arereleased upon degradation (bioerosion) of the construct.

In a preferred embodiment, this invention provides biodegradableconstructs which comprise a first PEA polymer in which A isL-phenylalanine (Phe-PEA) and a second PEA polymer in which A isL-leucine (Leu-PEA). Preferably, the ratio of Phe-PEA to Leu-PEA is from10:1 to 1:1; more preferably, the ratio of Phe-PEA to Leu-PEA is from5:1 to 2.5:1. The construct may be formed as a deformable sheet adaptedto conform to a biological surface.

In another embodiment, this invention provides bioerodable constructscomprising PEA polymers and further comprising a bioactive agent, whichmay be selected from the group consisting of antiseptics,anti-infectives, such as bacteriophages, antibiotics, antibacterials,antiprotozoal agents, and antiviral agents, analgesics,anti-inflammatory agents including steroids and non-steroidalanti-inflammatory agents including COX-2 inhibitors, anti-neoplasticagents, contraceptives, CNS active drugs, hormones, and vaccines.

In yet another embodiment, the bioerodable construct of this inventioncomprises an enzyme capable of hydrolytically cleaving the PEA polymer,such as α-chymotrypsin. In a preferred embodiment, the enzyme isadsorbed on the surface of the construct. In a particularly preferredembodiment, the construct contains bacteriophage which are released byaction of the enzyme.

This invention also provides a method of treating a patient having anulcerative wound comprising inserting into the wound or covering thewound with a bioerodable construct according to claim 1, wherein thebioerodable construct contains a bioactive agent, which may bebacteriophage, an antibiotic, an antiseptic, or an analgesic. The woundtreated by this invention may be open or infected, and the construct maybe in the form of a deformable sheet. In a preferred embodiment, theconstruct used in treatment of the wound contains bacteriophage specificfor bacteria found in the wound. The construct may also comprise anenzyme capable of hydrolytically cleaving the PEA polymer.

There is no currently available biodegradable polymer or polymeric blendcomposed entirely of naturally occurring and nontoxic building blocksshowing high plasticity (e.g., pliability when hydrated) together withhigh enzyme-catalyzed biodegradation rates, solubility in common organicsolvents like chloroform, and suitable for either impregnation or thespontaneous surface immobilization (adsorption) of the enzymes liketrypsin, a-chymotrypsin, and lipase. The polymeric blends of thisinvention provide all of these properties, permitting their use asmatrices for wound dressing/healing devices which are plastic and act torelease bioactive substances in a sustained/controlled fashion.

BRIEF DESCRIPTION OF THE FIGURE

The FIGURE shows lipase catalyzed biodegradation of polymers in vivoover a six month period.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The use of a bacteriophage lysate in the treatment of suppurativelesions that are inflamed or infected requires multiple and frequentapplications (e.g., 3-5 times a day) which increases consumption of boththe bacteriophage preparation and the wound dressings. From this pointof view the application of a bacteriophage reservoir, which provides forcontrolled release and prolonged action, is superior.

Bioresorbable (or bioerodable) polymers are the most appropriatematrices for preparing reservoirs of bacteriophages and/or otherbioactive compounds. Bioactive composites based on bioerodable polymersare known for controlled release of drugs to provide desirableconcentrations of bioactive substances in surrounding tissues.Compositites Composites made of bioerodable polymers disappear over timein a biological environment as the substance of the composite is egradeddegraded or dissolved by action of the surrounding biologic milieu. Thisdegradation may be facilitated by enzymes which catalyze cleavage ofcovalent bonds in the polymer. (Such enzymes may be present in thebilologic mileu biologic milieu or may be added exogenously, whether aspart of the construct or otherwise.) Controlled or sustained release ofa biologically active substance from a bioerodable construct refers to adelay in the dispersion of the biologically active substance relative tosimple diffusion from its point of introduction into the biologicalenvironment. Controlled release is generally due to some factor whichinterferes with normal diffusion of the substance, such as a diffusionbarrier or limited solubility of the diffusion substance. Thebioerodable constructs of this invention present a diffusion barrierwhich is removed progressively as the polymer degrades.

More recently, it has also been established that the rapid release ofpolymer degradation products in a sufficient amount into the surroundingtissues activates macrophages for the production of growth factors,which may accelerate wound healing. It is beneficial for polymericdegradation products to be either normal metabolic components or easilydigestible by cells. Polymers used as matrices should be plastic enoughto tightly cover wounds. It is also highly desirable for the polymericmatrix to be able either to immobilize enzymes (e.g. trypsin,alpha-chymotrypsin, lipase, etc.) on the surface by a simple method orincorporate them in the bulk matrix. These enzymes can participate inthe wound healing processes and can also erode polymers (e.g., bycatalyzing the hydrolysis of ester bonds in the polymeric backbone) witha constant and desirable rate to provided for the release ofbactericidal compounds as well as sufficient matrix degradation productsin the surrounding tissue to stimulate macrophages.

The inventor has synthesized new biodegradable poly (ester-amide)s(PEAs) composed of naturally occurring alpha-amino acids, includingessential ones like L-phenylalanine and L-leucine, and nontoxiccompounds like aliphatic and dicarboxylic acids. Suitable syntheticmethods are reported in Arabuli, et al. (1994), “Heterochain Polymersbased on Natural Amino Acids. Synthesis and enzymatic hydrolysis ofregular poly(ester-amide)s based on bis-(L-phenylalanine)alpha,omega-alkylene diesters and adipic acid,” Macromol. Chem. Phys.,195(6):2279, and Katsarava, et al. (1998) “Amino Acid Based BioanalogousPolymers. Synthesis and study of regular poly(ester-amide)s based onbis-(α-amino acid) α,ω-alkylene diesters and aliphatic dicarboxylicacid”, J. Polym. Sci.: Part A: Chemistry, 37:391-407, the entirety ofwhich are incorporated herein by reference. These rapidly bioresorbable,biocompatible poly(ester-amide)s may be used to form a bioerodablepolymer matrix.

The poly(ester-amides) of this invention do not contain any toxiccomponents. Alpha-amino acids, such as the essential amino acidsL-phenylalanine and L-leucine, are naturally-occurring products. Thesenormal metabolic components, upon release through biodegradation, aredigested by cells. Fatty acids and diols are well known nontoxicproducts commonly used in the food industry. They are also used asbuilding blocks for other classes of biodegradable polymers like polyanhydrides and poly-(ortho-ester)s approved by the U.S. Food and DrugAdministration (FDA) for clinical trials and other practicalapplications.

It is very important that the poly(ester-amide)s used in this inventionare soluble in organic solvents that do into inactivate bioactivecompounds such as bacteriophages. These polymers are soluble inchloroform in which the enzymes like trypsin, α-chymotrypsin, lipase aresufficiently stable for enzyme activity to survive the process ofpreparing enzyme-containing polymer constructs.

Enzymes can be added to polymeric solutions in chloroform in order toform enzyme-containing polymeric matrices when the solution is cast ontoglass plates and the solvent is evaporated. For polymeric filmsimpregnated by enzymes according to this method, the enzymes catalyzethe hydrolysis (erosion) of PEAs, which is important for the release ofbioactive substances into the surrounding tissues. The biodegradationrates of PEAs can vary over a wide range, spanning, e.g., 10¹-10³ mg/cm₂h. The degradation rate is a function of the enzyme activity in thecomposite. These polymers may be designed to release sufficient matrixdegradation products (polymeric debris) over time to activatemacrophages.

Enzymes may be spontaneously immobilized onto the surface of PEAs basedon L-phenylalanine through the simple immersion of the polymeric filmsin aqueous enzyme solution for varying lengths of time. (Immersion for,e.g., for 15-20 min is typical.) PEAs based on L-leucine do not readilyabsorb enzymes using this simple method, and thus, PEAs based onL-phenylalanine are more suitable for preparing biodegradable matriceswith surface-immobilized enzymes. However, PEAs based on L-phenylalaninedo not possess sufficient plasticity for use as wound coverings. PEAscomposed of L-leucine are pliable when hydrated (i.e., water acts as aplasticizer) and more suitable for biological applications such as woundcoverings (dressings); however the films prepared from L-leucine PEAsare very sticky, adhering to themselves, and inconvenient to work with.In addition, L-leucine based PEAs immobilize enzymes poorly.

The present invention has discovered that the detrimentalcharacteristics inherent in each class of PEAs can be overcome byblending them. Polymeric blends prepared from approximately 70% ofL-phenylalanine based PEAs and 30% of L-leucine based PEAs showed:

-   -   good plasticity (necessary to cover wounds tightly),    -   lack of self-adhesion, and    -   ability to immobilize enzymes.

As contemplated by the present invention, the polymer blend which is thebasis for the invention has sufficient plasticity to permit a film madewith the polymer blend to be manually deformed to fit tightly to anirregular biological surface (e.g., a concave wound surface).Additionally, films made with the polymer blend are readily separable bygentle manual force, leaving each sheet of film intact upon separation.Finally, the surface of an object made with the polymer blend of thisinvention will absorb proteins, such that measurable enzyme activity canbe detected adhered to the surface of the object after it is dipped intoa solution of the enzyme.

This invention provides polymer blends comprising at least two PEAs offormula II. Preferably the blend contains one PEA in which R correspondsto the side chain of phenylalanine (Phe-PEA) and one PEA in which Rcorresponds to the side chain of leucine (Leu-PEA). The ratio of Phe-PEAto Leu-PEA may vary from 10:1 to 1:1, but is preferably from 5:1 to2.5:1. Other PEAs (and indeed other polymers) may be included in theblend, so long as the resultant blend still exhibits the desiredproperties described above. The other polymers in the blend will, ofcourse, be soluble in the solvent in which the blend is dispersed forpreparing the constructs according to this invention. Leu-PEA andPhe-PEA are soluble in polar organic solvents includingdimethyl-formamide (DMF), dimethylacetamide, dimethylsulfoxide (DMSO),trifluoroethanol, hexafluoroisopropanol and the like, or neutral organicsolvents including chloroform and the like. Chloroform and similarsolvents are preferred for preparation of bioerodable films containingbioactive components due to greater volatility (important for preparingfilms) and reduced tendency to inactivate enzymes (such as chymotrypsinor lipase), bacteriophages or other bioactive components.

In a preferred mode, the polymer blend of this invention is formed intoa bioerodable film. The films of this invention may be a single layer ormultiple layers, such as a bilayer film having one layer of a PEA blendand an adjacent layer of poly(siloxane elastomer). However, alternativebioerodable constructs using the polymer blend are easily within theskill of the art and within the contemplation of this invention. Forexample, the polymer blend may be used to provide a bioerodable coatingon a support material which may or may not be biodegradable, such as afibrous or non-fibrous three-dimensional construct or a woven support.Suitable forms for the three-dimensional constrcts constructs of thisinvention are foams, which may be formed by conventional means. Forexample, Phe-PEA/Leu-PEA blends can be prepared as foams as follows: asuspension of bacteriophages and other bioactive substanses substances(about 1 g) in the solution of Phe-PEA/Leu-PEA blend (1 g) in chloroform(10 mL) can be cast onto hydrophobic surface and 90-99% of chloroformevaporated at r.t. under atmospheric pressure. Afterwards a reducedpressure may be applied at room temperature to remove residualchloroform, and the resulting foamed film dried for 12 h under reducedpressure. According to another procedure 1-10% (of chloroform volume) ofn-pentane may be added to the suspension above. The mixture may be castonto a hydrophobic surface and allowed to dry at room temperature for 24h, and the foamed film may be subjected to a final drying under reducedpressure for 12 h. Foamed films may also be obtained using ultrasonicdisintegration techniques.

Constructs prepared with the polymers of this invention may be part ofdevices including a support material to be used as, for example,bandages for wounds or burn dressings. Of course, the blends forming acoating on a woven support will preferably retain the flexibility and/orelasticity of blends used for film-forming, but a blend for coating arigid, three-dimensional construct may be less elastic. Such blends mayhave higher Phe-PEA content, and coatings in which Phe-PEA is the onlyPEA polymer are within the contemplation of this invention for suchapplications.

In another mode, this invention contemplates constructs consisting allor in part of a blend according to this invention which may besurgically implanted. Constructs according to this invention may also beformed into devices for wound packing, such as gel foams, or may be usedas components in surgical appliances, such as Penrose drains, indwellingcatheters, catheters for peritoneal dialysis, and any other appliancesthat are in contact with body cavities, the blood circulation, or thelymphatic circulation and are either used to treat potential infectionsor are at risk of becoming infected. This invention also contemplatesappliances for oral hygiene, including gum implants (e.g., forperiodontal disease or dental caries). Such constructs will preferablycontain bioactive material released in a controlled manner upon erosionof the construct. Suitable selections of particular bioactive inclusionswill be readily apparent to the skilled artisan in view of the intendedsite of implantation. For example, composites containing bactericidalagenst agents such as bacteriophage may be implanted in the body totreat osteomyelitis, etc. Alternatively, bioerodable composites of thisinvention could be used for sustained/controlled release of anticancerand/or other drugs at a target site. Bioactive materials may be releasedin a controlled fashion by diffusion from within the construct, or bydegradation of the construct, or by a combination of these processes.

Bioactive and/or inactive biocompatible materials may be included in theerodable construct in amounts up to 60% or more by weight, so long astheir inclusion does not destroy the desirable properties of filmsaccording to this invention. Bioactive materials contemplated forinclusion in the bioerodable constructs of this invention include, butare not limited to, antiseptics, anti-infectives, such asbacteriophages, antibiotics, antibacterials, antiprotozoal agents, andantiviral agents, analgesics, anti-inflammatory agents includingsteroids and non-steroidal anti-inflammatory agents including COX-2inhibitors, anti-neoplastic agents, contraceptives, CNS active drugs,hormones, and vaccines. In particular, constructs may include one ormore of calcium gluconate and other phage stabilizing additives,hyaluronidase, fibrinolysine and other fibrinolytic enzymes,methyluracyl and other agents stimulating metabolic processes, sodiumhydrocarbonate, L-arginine and other vasodilators, Benzocaine and otherpain killers, mono- and disaccharides, polysaccharides andmucopolysaccharides, Metronidazol and other anti-protozoa drugs,Clotrimazolum and other anti-fungal drugs, thrombine and otherhemostatics, vitamins, Prednizolone and other anti-inflammatorysteroids, and Voltaren (Sodium diclofenac) and other anti-inflammatorynon-steroid drugs. Of course the skilled artisan will in any caseconfirm that particular construct formulations retain the desiredproperties as discussed herein, and constructs which exhibit none ofthese properties are outside the contemplation of this invention.

In one preferred mode, this invention provides a novel approach tomanagement of poorly healing and poorly vascularized wounds (which mayinclude diabetic foot ulcers, pressure ulcers in patients with reducedmobility, and other ulcers and open skin lesions lesions). In medicine,poorly healing wounds, such as those seen in diabetic patients with footulcers, and in bedridden patients with pressure sores, represent a majorand very expensive management problem. Use of antibiotics in thissetting is generally not efficacious. Because of poor vascularization,antibiotics seldom achieve therapeutic levels in affected areassufficient to eradicate infection. Moreover, because of the recurrentcourses of antibiotics that such patients have often received, thebacterial pathogens causing the infections are often antibioticresistant. In this mode, as well as other wound treatment embodiments,the controlled-release character of the polymer constructs according tothis invention avoid the necessity of constant re-application ofbactericidal material, as well as the need for associated dressingchanges.

Biocomposites mediating a sustained/controlled release of appropriatetherapeutic agents have proven to be especially efficacious for healinginfected wounds and cavities. Film materials, so called “artificialskin”, prepared from these biocomposites have important therapeuticeffects:

-   -   Polymer material, when applied to the surface of such wounds,        acts as a protector from external mechanical actions and        bacterial invasion, and further prevents heat and moisture loss        that occur as a result of uncontrolled water evaporation from        the injured surface; and    -   The slow-release properties of the biologically-active compound        can be exploited to promote appropriate, steady release of        anti-bacterial agents at the site of infection.

Use of biocomposite “artificial skin” does not require patientimmobilization, and thereby facilitates a return to daily lifeactivities, an important consideration in this class of patients.

A key element in the management of chronically infected wounds is thesuppression of pathogenic bacterial flora. With biocomposite materials,this can be achieved by introducing bacteriocidal substances into thebiocomposite structure. Antibiotics may be used in this setting, buttheir efficacy is increasingly limited by the development of antibioticresistance. More recently, there has been interest in the introductioninto biocomposites of such bactericidal substances as silversulfadiazine (and related diazine derivatives of sulfanilamide), furagin(and pharmaceutically acceptable salts thereof) and chlorohexydine (andpharmaceutically acceptable salts thereof). However, utilization of suchcompounds may be limited by their inherent toxicity, particularly forpatients with underlying kidney or liver disease.

Incorporation of bacteriophages into such biocomposite materialsprovides an alternative approach. Bacteriophage are viruses that killspecific bacteria. The lysis of microorganisms by viruses was discoveredat the beginning of the 20th century. Any one phage tends to be highlyspecific for certain bacteria, requiring that therapy be carefullytargeted (i.e., there is no analogy to the broad-spectrum antibioticswhich can “kill everything”). However, this also means that phagetherapy can be used to kill specific pathogens without disturbing normalbacterial flora.

Phages have been reported to be effective in treating skin infectionscaused by Pseudomonas, Staphylococcus, Klebsiella, Proteus, E. coli, andother pathogenic species; success rates in these studies have rangedfrom 75 to 100%, depending on the pathogen. However, for these studiesbacteriophages were introduced in a variety of vehicles: aqueous liquidpreparations, aerosols and creams.

The polymeric blend composed of L-phenylalanine, L-leucine, adipic acid,and butane-diol-1,4 has been successfully used for preparing bioactivecomposites containing bactericidal substances. The wound dressingsobtained based on this biocomposite material showed high wound healingproperties.

Starting from the materials mentioned above it seems that bioactivecomposite based on bioresorbable (bioerodable) polymer and containing acomplex of bacteriophages as a bactericidal substance will be aneffective dressing material with accelerated wound healing ability.Selection of suitable bacteriophage is described in U.S. ProvisionalPatent Application No. 60/175,415, entitled “Bacteriophage Specific forVancomycin Resistant Enterococci (VRE)”, filed Jan. 11, 2000, and U.S.Provisional Patent Application Nos. 60/175, 416, filed Jan. 11, 2000,and 60/205,240, filed May 19, 2000, both entitled “Method And Device ForSanitation Using A Bacteriophage”, the disclosures of which areincorporated by reference in their entireties.

EXAMPLE

A complex of polyvalent bacteriophages directed toward Staphylococcusspecies, Streptococcus species, E. coli, Proteus species, andPseudomonas aeruginosa with a titer of 2×10⁶-2×10⁷ plaque-forming units,was prepared and used as bioactive substance for this study.Bacteriophage were prepared as a lyophilized dry powder as follows:bacteriophages suspended in an aqueous sucrose-gelatin mixture werelyophilized, resulting in a dry mass that was ground into fine powder.In this process, 50 mg of dry preparation corresponds to 1 ml of liquidbacteriophage with a titer of 2×10⁶-2×10⁷. None of the individualcomponents of bioactive composites (polymer, organic solvent,alpha-chymotrypsin, lipase) affected bacteriophages activity—100% ofstarting activity was retained in all cases.

A bioactive film was prepared as follows: A fine suspension of drybacteriophage in a polymer solution with an appropriate solvent was caston a glass surface and dried to constant weight. A composite wasobtained in the from of a film with the following characteristics: mass1 g, film area—65-65 cm², thickness—0.2-0.3 mm. Afterwardsalpha-chymotrypsin was immobilized on the surface of the film.Optionally, the film was perforated. For particular applications,analgesics and/or antibiotics were added to the composite as well.

The activity of the resultant film in in vitro experiments wasdetermined using a bacterial lawn on solid media. Activity was estimatedby measuring the width of the zone of lysis. The activity of the filmcoincides with the activity of dry bacteriophages used; pure polymericfilm did not reveal any bactericidal activity.

The kinetics of bacteriophage release from 9 cm disks of the film wasstudied in phosphate buffer under physiological conditions (see Table1). One can see that release of bacteriophages during first 24 hoursboth from α-chymotrypsin-immobilized and α-chymotrypsin-free films wascomparable; for enzyme-immobilized film it was only 1.5-2 times higher.This can be explained by extensive desorption of bacteriophages from thesurface zone of enzyme free film. However, when the films weretransferred to fresh buffer at 24 hours and 120 hours, theenzyme-catalyzed erosion mechanism became important at later stages forreleasing bacteriophages from the bulk of the film, and difference inrelease rate reached more than one order in magnitude. Clearly,alpha-chymotrypsin promotes the release of bacteriophages from bioactivecomposite.

TABLE Sustained Release of Bacteriophages and Antibiotics from MedicatedWound Covering Film Release of bacteriophages from 9 cm dia. Phe-PEAfilm disks into 10 mL of Phosphate buffer 0.2 M, pH 7.4, T = 37° C. A 9cm Phe-PEA/bacterio- phage film disk contains approximately 1800 × 10⁴bacteriophages. Titer of bacteriophages in 1 mL solution CompositeComposite bacteriophage/Phe-PEA film Time in bacteriophage/Phe-PEAwithout surface-immobilized hours film with α-chymotrypsinα-chymotrypsin 1 2.0 × 10⁴ 1.3 × 10⁴ 3 5.0 × 10⁴ 3.0 × 10⁴ 24 8.0 × 10⁴4.0 × 10⁴

24 h later, after transfer to a new 10 mL portion of the buffer

1 3.2 × 10⁴ 1.3 × 10⁴ 3 9.0 × 10⁴ 3.1 × 10⁴ 96 200.0 × 10⁴  90.0 × 10⁴ 

120 h later, after transfer to a new 10 mL portion of the buffer

Composite Composite bacteriophage/Phe-PEA film Time inbacteriophage/Phe-PEA without surface-immobilized hours film withα-chymotrypsin α-chymotrypsin 1 2.5 × 10⁴ 0.06 × 10⁴ 4 5.0 × 10⁴ 0.20 ×10⁴

It should be noted that surface immobilized α-chymotrypsin can play anadditional role namely it can decompose both peptides and denaturatedproteins. this enzymatic debridment, as it is known from literature,leads to the sanitation of a wound and accelerates healing.

The activity of films according to this invention was checkedperiodically for 1.5 years against both preexisting laboratory strainsand newly received bacterial strains, and the film retained activityover this period. The surface immobilized enzyme was active for thisperiod as well. The FIGURE shows lipase catalyzed biodegradation ofpolymers in vivo over a six month period. The in vivo data is summarizedin Table 2.

TABLE 2 In vivo Degradation of Biocomposites Number Number of films ofper one Duration Sample rats rat (days) Result 4-L-Phe-4 2 2 109 Filmswere completely absorbed, in one case a trace of connective tissuecapsule was observed. 4-L-Phe-4 2 2 123 Films were completely absorbed,no trace of tissue reaction was observed 4-L-Phe-4 2 2 175 Films werecompletely absorbed, no trace of tissue reaction was observed.4-L-Phe-4-Lip 3 2 39 In 2 rats films were completely absorbed, in onerat both films were incapsulated*encapsulated*. 4-L-Phe-4-Lip 1 2 42Films were completely absorbed, no trace of tissue reaction wasobserved. 4-L-Phe-4-Lip 4 4 44 Films were completely absorbed, no traceof tissue reaction was observed. 4-L-Phe-4-Lip 1 2 45 Both films wereincapsulated* encapsulated*. 4-L-Phe-4-Lip 5 3 77 14 films werecompletely absorbed, only one film was incapsulated*encapsulated*.4-L-Phe-4-Lip 2 2 145 Films were completely absorbed, no trace of tissuereaction was observed. *In these cases lipase was found to beinactivated, that is it did not catalyze the hydrolysis of poly(esteramide). Totally 57 films (each 20-25 mg) were implanted subcutaneouslyto into rats, 52 films were completely absorbed. Only 5 films of4-L-Phe-4-Lip series were incapsulated encapsulated owing to enzymeinactivation. 4-L-Phe-4-PEA based on L-phenylalanine, adipic acid, andbutanediol-1,4,4-L-Phe-4-Lip - the same, lipase impregnated (10 mglipase per 1 g of PEA).

1. A bioerodable construct for controlled release of bioactivematerials, said construct comprising a blend of at least twopoly(ester-amide) polymers (PEA) prepared by polymerizing a diol (D),wherein the diol (D) is not bisphenol; a dicarboxylic acid (C), whereinthe dicarboxylic acid (C) is not phthalic acid; and an alpha-amino acid(A) through ester and amide links in the form (DACA)_(n). , wherein thePEA polymer has the formula:

wherein k=2-12, m=2-12, and R=CH₂CH(CH₃)₂, or CH₂C₆H₅. and, wherein theblend comprises a first PEA polymer in which A is phenylalanine(Phe-PEA) and a second PEA polymer in which A is leucine (Leu-PEA) at aratio of Phe-PEA to Leu-PEA is from of 10:1 to 1:1.
 2. The construct ofclaim 1, wherein k=2, 3, 4, and 6 and m=4 or
 8. 3. The construct ofclaim 1, wherein the ratio of Phe-PEA to Leu-PEA is 5:1 to 2.5:1.
 4. Theconstruct according to any one of claims 1,2 or 3, wherein the constructis a deformable sheet adapted to conform to a biological surface.
 5. Theconstruct according to claim 4, further comprising a bioactive agent. 6.The construct of claim 5, wherein the bioactive agent is selected fromthe group consisting of antiseptics, anti-infectives, such asbacteriophages, antibiotics, antibacterials, antiprotozoal agents, andantiviral agents, analgesics, anti-inflammatory agents includingselected from the group consisting of steroids and non-steroidalanti-inflammatory agents including selected from the group consisting ofCOX-2 inhibitors, anti-neoplastic agents, contraceptives, CNS activedrugs, hormones, and vaccines.
 7. The construct according to claim 5,wherein the construct comprises an enzyme capable of hydrolyticallycleaving the PEA polymer.
 8. The construct according to claim 7, whereinthe enzyme is α-chymotrypsin.
 9. The construct according to claim 7,wherein the enzyme is adsorbed on the surface of the construct.
 10. Theconstruct according to claim 7, wherein the construct contains abacteriophage which are is released by action of the enzyme.
 11. Amethod of treating a patient having an ulcerative wound comprisinginserting into the wound or covering the wound with a bioerodableconstruct according to claim 1, wherein the bioerodable construct is adeformable sheet containing a bioactive agent.
 12. The method of claim11, wherein the bioactive agent is comprises a bacteriophage, anantibiotic, an antiseptic, or an analgesic.
 13. The method of claim 11,wherein the wound is open or infected.
 14. The method according to claim12, wherein the bacteriophage are specific for bacteria found in thewound.
 15. The method according to any one of claim claims 11-14,wherein the construct also comprises an enzyme capable of hydrolyticallyclearing the PEA polymer.
 16. The construct according to any one ofclaims 1, 2 or 3, further comprising a bioactive agent.
 17. Theconstruct of claim 16, wherein the bioactive agent is selected from thegroup consisting of antiseptics, anti-infectives, such asbacteriophages, antibiotics, antibacterials, antiprotozoal agents, andantiviral agents, analgesics, anti-inflammatory agents includingselected from the group consisting of steroids and non-steroidalanti-inflammatory agents including selected from the group consisting ofCOX-2 inhibitors, anti-neoplastic agents, contraceptives, CNS activedrugs, hormones, and vaccines.
 18. The construct according to any one ofclaims 1, 2 or 3, wherein the construct comprises an enzyme capable ofhydrolytically cleaving the PEA polymer.
 19. The construct according toclaim 18, wherein the enzyme is α-chymotrypsin.
 20. The constructaccording to claim 18, wherein the enzyme is adsorbed on the surface ofthe construct.
 21. The construct according to claim 18, wherein theconstruct contains a bacteriophage which are is released by action ofthe enzyme.
 22. The construct according to claim 1 wherein the blend isformed into a bioerodable coating on a support material or is formedinto a bioerodable film.
 23. The construct according to claim 22 whereinthe blend is formed into a bioerodable coating comprising bioactivematerial.
 24. The construct according to claim 1 wherein the constructis a device that can be surgically implanted.
 25. The constructaccording to claim 24 wherein the implantable device is an indwellingcatheter or appliance for oral hygiene.
 26. A bioerodable construct forcontrolled release of bioactive materials, said construct comprising ablend of at least two poly(ester-amide) polymers (PEA), wherein each PEApolymer has the formula:

wherein k=2-12, m=2-12, and R=CH(CH₃ ) ₂ , CH ₂ CH (CH ₃ ) ₂ , CH(CH ₃)CH ₂ CH ₃ , (CH ₂ ) ₃ CH ₃ , CH ₂ C ₆ H ₅ , or (CH ₂ ) ₂ SCH ₃ ,wherein the blend comprises a PEA polymer wherein R=CH₂ CH (CH ₃ ) ₂ ora PEA polymer wherein R=CH ₂ C ₆ H ₅.
 27. The construct according toclaim 26, wherein the blend comprises a first PEA polymer wherein R=CH₂CH(CH ₃ ) ₂ , and a second PEA polymer wherein R=CH ₂ C ₆ H ₅.
 28. Theconstruct of claim 27, wherein the first PEA polymer and second PEApolymer are present in a ratio of 10:1 to 1:1.
 29. The construct ofclaim 28, wherein the ratio of first PEA polymer to second PEA polymeris 5:1 to 2.5:1.
 30. The construct of claim 26, wherein k=2, 3, 4, or 6and m=4 or
 8. 31. The construct according to claim 26, wherein theconstruct is deformable sheet adapted to conform to a biologicalsurface.
 32. The construct according to claim 31, further comprising abioactive agent.
 33. The construct of claim 32, wherein the bioactiveagent is selected from the group consisting of antiseptics,anti-infectives, bacteriophages, antibiotics, antibacterials,antiprotozoal agents, and antiviral agents, analgesics,anti-inflammatory agents selected from the group consisting of steroidsand non-steroidal anti-inflammatory agents selected from the groupconsisting of COX-2 inhibitors, anti-neoplastic agents, contraceptives,CNS active drugs, hormones, and vaccines.
 34. The construct according toclaim 32, wherein the construct comprises an enzyme capable ofhydrolytically cleaving the PEA polymer.
 35. The construct according toclaim 34, wherein the enzyme is α-chymotrypsin.
 36. The constructaccording to claim 34, wherein the enzyme is adsorbed on the surface ofthe construct.
 37. The construct according to claim 34, wherein theconstruct contains a bacteriophage which is released by action of theenzyme.
 38. A method of treating a patient having an ulcerative woundcomprising inserting into the wound or covering the wound with abioerodable construct according to claim 26, wherein the bioerodableconstruct is a deformable sheet containing a bioactive agent.
 39. Themethod of claim 38, wherein the bioactive agent comprises abacteriophage, an antibiotic, an antiseptic, or an analgesic.
 40. Themethod of claim 38, wherein the wound is open or infected.
 41. Themethod according to claim 39, wherein the bacteriophage is specific forbacteria found in the wound.
 42. The method of claim 38, wherein theconstruct also comprises an enzyme capable of hydrolytically cleavingthe PEA polymer.
 43. The construct according to claim 26 furthercomprising a bioactive agent.
 44. The construct of claim 43, wherein thebioactive agent is selected from the group consisting of antiseptics,anti-infectives, bacteriophages, antibiotics, antibacterials,antiprotozoal agents, and antiviral agents, analgesics,anti-inflammatory agents selected from the group consisting of steroidsand non-steroidal anti-inflammatory agents selected from the groupconsisting of COX-2 inhibitors, anti-neoplastic agents, contraceptives,CNS active drugs, hormones, and vaccines.
 45. The construct according toclaim 26, wherein the construct comprises an enzyme capable ofhydrolytically cleaving the PEA polymer.
 46. The construct according toclaim 45, wherein the enzyme is α-chymotrypsin.
 47. The constructaccording to claim 45, wherein the enzyme is adsorbed on the surface ofthe construct.
 48. The construct according to claim 45, wherein theconstruct contains a bacteriophage which is released by action of theenzyme.
 49. The construct according to claim 26 wherein the blend isformed into a bioerodable coating on a support material or is formedinto a bioerodable film.
 50. The construct according to claim 49 whereinthe blend is formed into a bioerodable coating comprising bioactivematerial.
 51. The construct according to claim 26 wherein the constructis a device that can be surgically implanted.
 52. The constructaccording to claim 51 wherein the implantable device is an indwellingcatheter or appliance for oral hygiene.
 53. A bioerodable construct forcontrolled release of bioactive materials, said construct comprising ablend of at least two poly(ester-amide) polymers (PEA) prepared bypolymerizing a diol (D), wherein the diol (D) is not bisphenol; adicarboxylic acid (C), wherein the dicarboxylic acid (C) is not phthalicacid; and an alpha-amino acid (A) through ester and amide links in theform (DACA)_(n) , wherein the alpha-amino acid (A) of each PEA polymerof the blend is selected from the group of amino acids having aliphaticside chains, amino acids having sulfur-containing side chains, and aminoacids having side chains containing aromatic rings, and wherein thealpha-amino acid of at least one of the PEA polymers is phenylalanine orleucine.
 54. The construct of claim 53 wherein the amino acid havingaliphatic side chains is selected from valine, leucine, isoleucine, andnorleucine.
 55. The construct of claim 53, wherein the amino acid havingsulfur-containing side chains is methionine.
 56. The construct of claim53, wherein the amino acid having side chains containing aromatic ringsis phenylalanine.
 57. The construct of claim 53 the blend comprises afirst PEA polymer in which A is phenylalanine and a second PEA polymerin which A is leucine.
 58. A bioerodable construct for controlledrelease of bioactive materials, said construct comprising a blend of atleast two poly(ester-amide) polymers (PEA), wherein each PEA polymer hasthe formula:

wherein k=2-12, m=2-12, wherein R represents an amino acid side chain,and the amino acid side chain is selected from aliphatic side chains,sulfur-containing side chains, and side chains containing aromaticrings, and wherein the blend comprises a PEA polymer wherein R is anamino acid side chain containing an aromatic ring or a PEA polymerwherein R is an aliphatic amino acid side chain.
 59. The construct ofclaim 58 wherein the aliphatic amino acid side chain is selected fromvaline, leucine, isoleucine, and norleucine side chains.
 60. Theconstruct of claim 58, wherein the sulfur-containing side chain is amethionine side chain.
 61. The construct of claim 58, wherein the sidechain containing aromatic rings is a phenylalanine side chain.
 62. Theconstruct of claim 58 the blend comprises a first PEA polymer in which Ris a phenylalanine side chain, and a second PEA polymer in which R is aleucine side chain.
 63. The construct according to claim 1 wherein theconstruct is a device for wound packing.
 64. The construct according toclaim 63 wherein the construct is a foam.
 65. The construct according toclaim 53 wherein the construct is a device for wound packing.
 66. Theconstruct according to claim 65 wherein the construct is a foam.